CN105727262B

CN105727262B - Application of pancreatic regeneration source protein in preparation of medicine for promoting excessive inflammation wound healing

Info

Publication number: CN105727262B
Application number: CN201410747546.3A
Authority: CN
Inventors: 赖玉平; 吴叶林; 刘媛琪; 全艳春; 鲁纪龙; 雷虎
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2014-12-08
Filing date: 2014-12-08
Publication date: 2020-08-25
Anticipated expiration: 2034-12-08
Also published as: CN105727262A

Abstract

The invention discloses application of pancreatic regeneration source protein in preparation of a medicine for promoting excessive inflammatory wound healing, wherein the pancreatic regeneration source protein is locally applied, so that excessive inflammatory reaction of skin wounds can be effectively inhibited, and wound healing is promoted.

Description

Application of pancreatic regeneration source protein in preparation of medicine for promoting excessive inflammation wound healing

Technical Field

The invention relates to application of pancreatic regeneration source protein or derivatives thereof in preparing a medicament for promoting excessive inflammatory wound healing.

Background

Inflammation is a defensive response of the body to infection, irritation or injury, and is generally beneficial to the human body and helps the body to eliminate necrotic or infected cells, etc. resulting from the irritation or injury. However, excessive inflammatory response can damage body tissues, resulting in diseases such as rheumatoid arthritis, psoriasis, wound disunion, etc. Excessive inflammation is one of the major causes of non-healing of skin wounds, particularly diabetic skin wounds. Excessive inflammatory reaction causes the reduction of the proliferation and migration capacity of skin keratinocytes and fibroblasts, and the increase of apoptosis of cells, thereby inhibiting the normal healing of wounds. Difficulties in diabetic wound healing have been the leading cause of the highest hospital admissions among diabetic patients. Of the diabetic patients, 15% have symptoms of non-healing wounds, while 84% of these patients are at risk of amputation due to non-healing wounds. Therefore, the finding of the medicine and the method for inhibiting the diabetes wound inflammation have important significance for the treatment of the diabetes wound.

The regeneration islet-derived protein (Reg) belongs to the C-type lectin family, and comprises an N-terminal signal peptide and a C-type lectin domain which is Zn2+ dependent and not Ca2 +. The human REG3A (encoded by REG3 a) protein belongs to one of them, and the corresponding one is the murine RegIII γ protein (encoded by Reg3 g), which have high homology. REG3A/RegIII gamma protein was originally found to be produced upon pancreatic injury, has the ability to promote pancreatic regeneration, and promotes the proliferation of islet cells after pancreatic injury, and REG3A was later found to prevent hepatocyte apoptosis in acute hepatic failure, and thus is expected to be one of the drugs for treating acute hepatic failure. The Reg protein can also be used as an antibacterial protein for controlling the diffusion of intestinal flora and preventing pathogenic bacteria from infecting the intestinal tract.

The inventor laboratory reports REG3A/RegIII gamma for the first time in 2012 that REG3A/RegIII gamma is generated when skin is damaged, and can promote the proliferation of skin keratinocytes by inhibiting the differentiation of the keratinocytes so as to regulate the healing of normal wounds, thus opening up a new field of research of Reg protein on skin wounds.

Disclosure of Invention

One of the purposes of the invention is to provide a new application of pancreatic regeneration source protein, wherein the pancreatic regeneration source protein can effectively inhibit excessive inflammatory reaction of skin wounds and promote the healing of wounds, particularly diabetic wounds.

The invention also aims to provide a novel pancreatic regeneration source protein derivative and application thereof in preparing medicaments for inhibiting inflammatory reaction, promoting wound healing and resisting infection.

In the invention, the pancreas regeneration source protein comprises human pancreas regeneration source protein REG3A shown in SEQ ID NO.1 and mouse pancreas regeneration source protein RegIII gamma shown in SEQ ID NO. 2.

Wherein, the human pancreas regeneration origin protein REG3A contains 175 amino acids, the mature peptide is 149 amino acids (27 th to 175 th amino acids), and the sequence is shown in SEQ ID NO. 1:

EEPQRELPSARIRCPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKFTD(SEQ ID NO.1)

the mouse pancreas regeneration source protein RegIII gamma contains 174 amino acids, the mature peptide is 148 amino acids (27 th to 174 th amino acids), and the sequence is shown as SEQ ID NO. 2:

EVAKKDAPSSRSSCPKGSRAYGSYCYALFSVSKNWYDADMACQKRPSGHLVSVLSGAEASFLSSMIKSSGNSGQYVWIGLHDPTLGYEPNRGGWEWSNADVMNYINWETNPSSSSGNHCGTLSRASGFLKWRENYCNLELPYVCKFKA(SEQ ID NO.2)

the invention provides a pancreatic regeneration protein derivative, which comprises a pancreatic regeneration protein derivative C-REG3A (40-173) shown in SEQ ID NO.3 and a pancreatic regeneration protein derivative C-RegIII gamma (40-174) shown in SEQ ID NO. 4.

Wherein, the pancreas regeneration origin protein derivative C-REG3A (40-173), namely CTLD domain of human pancreas regeneration origin protein REG3A, is composed of 134 amino acids from 40 th to 173 th of REG3A, and the sequence is shown in SEQ NO. 3:

CPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKF(SEQ IDNO.3)

wherein, the pancreas regeneration source protein derivative C-RegIII gamma (40-174), namely the CTLD structural domain of the mouse regeneration source protein RegIII gamma, is composed of 135 amino acids from 40 th to 174 th of RegIII gamma, and the sequence is shown in SEQ ID NO. 4:

CPKGSRAYGSYCYALFSVSKNWYDADMACQKRPSGHLVSVLSGAEASFLSSMIKSSGNSGQYVWIGLHDPTLGYEPNRGGWEWSNADVMNYINWETNPSSSSGNHCGTLSRASGFLKWRENYCNLELPYVCKFKA(SEQ IDNO.4)

in the present invention, the pancreatic regeneration-derived protein may be of natural, synthetic, genetic recombinant, animal or human origin. The pancreatic regeneration-derived protein derivative can be synthesized or obtained by gene recombination.

The pancreatic-derived protein or its derivative can be prepared by conventional techniques, for example, by amplifying the desired gene by PCR, transforming a host cell of a suitable prokaryote, such as Escherichia coli, with an expression vector containing the gene sequence of REG3A/RegIII γ protein or its derivative to obtain a genetically engineered strain, and fermenting the genetically engineered strain to obtain the pancreatic-derived protein or its derivative.

The invention provides application of pancreatic regeneration source protein in preparation of a medicine for promoting excessive inflammation wound healing. The invention provides a method for preparing a medicament for inhibiting excessive inflammatory reaction of wounds and promoting diabetic wound healing by using the pancreatic regeneration source protein. Wherein the pancreas regeneration source protein comprises a human pancreas regeneration source protein REG3A shown in SEQ ID NO.1 and a mouse pancreas regeneration source protein RegIII gamma shown in SEQ ID NO. 2. Wherein the medicament utilizes the pancreatic regeneration source protein to inhibit excessive inflammatory reaction and promote wound healing. Wherein the wound is a chronic inflammatory wound, a pressure ulcer wound, a bedsore wound, a burn wound, a surgical wound, etc. Wherein the chronic inflammatory wound comprises a diabetic wound, a diabetic foot ulcer and the like.

In the present invention, the inflammation of skin wound refers to the inflammatory reaction of skin at the time of wound occurrence, and inflammation is a defense reaction of the body against stimulation, which is usually beneficial, but sometimes, the body is damaged by excessive inflammatory reaction.

In the invention, the excessive inflammation refers to that an organism cannot resist injury caused by inflammatory factors or infection in an inflammatory reaction, and the injury is usually expressed by continuous high expression or diffusion of the inflammatory factors such as TNF and IL-6, so that tissues are continuously injured, and the inflammatory process is not healed.

In the present invention, the excessive inflammatory wound may be a chronic inflammatory wound, a pressure ulcer wound, a decubitus wound, a burn wound, a surgical wound, or the like. Wherein the chronic inflammatory wound comprises a diabetic wound, a diabetic foot ulcer. Chronic inflammation is relatively acute inflammation, and inflammatory factors last for a long time, from months to years.

The invention also provides application of the pancreatic regeneration source protein in preparation of a medicament for treating diabetic wound healing. The invention also provides application of the pancreatic regeneration source protein in inhibiting excessive inflammation of diabetic skin wounds. The local application of the pancreatic regeneration source protein can reduce the inflammatory reaction around the diabetic wound and promote the wound healing. In a specific embodiment, the diabetes wound is injected with the pancreatic regeneration source protein, so that excessive inflammation of skin wound is inhibited, and wound healing is promoted.

The invention also provides application of the pancreatic regeneration protein or the derivative thereof in inhibiting the inflammation induced by the ligand Poly (I: C) of the Toll-like receptor 3, wherein the pancreatic regeneration protein and the pancreatic regeneration protein derivative can be used as inhibitors of inflammatory response induced by the Poly (I: C). The pancreatic regeneration source protein inhibits the expression of inflammatory factors TNF-alpha and IL-6 around wounds. The research shows that: the inflammatory response of non-infected wounds mainly depends on Toll-like receptor 3, while in the invention, the pancreatic regeneration protein or the derivative thereof inhibits the expression of human primary keratinocytes TNF-alpha and IL-6 induced by ligand poly (I: C) of Toll-like receptor 3, and the pancreatic regeneration protein or the derivative thereof has an inhibitory effect on wound inflammation proved from a cellular level.

The invention provides application of a pancreatic regeneration protein derivative in preparation of a medicament for inhibiting inflammatory reaction and promoting wound healing. The invention provides a method for inhibiting inflammatory reaction and promoting wound healing by using pancreatic regeneration source protein derivatives. In a specific embodiment, wound healing is promoted by injecting a derivative of a pancreatic regenerating source protein into the wound. Wherein the pancreatic regeneration-derived protein derivatives comprise human pancreatic regeneration-derived protein derivatives C-REG3A shown in SEQ ID NO.3 and murine pancreatic regeneration-derived protein C-RegIII gamma shown in SEQ ID NO. 4.

Wherein the wound comprises an infected wound or a non-infected wound; the non-infected wound comprises a normal wound, a chronic inflammatory wound, a pressure ulcer wound, a bedsore wound, a burn wound and a surgical wound; the chronic inflammatory wound comprises a diabetic wound and a diabetic foot ulcer.

The invention also provides application of the pancreatic regeneration source protein or the derivative thereof in cosmetics and skin care products as a wound healing promoter or a wound inflammation inhibitor or an anti-infective agent. The pancreas regeneration source protein or the derivative thereof can be used for preparing cosmetics, skin care products, daily necessities or medicines and the like. Wherein the pancreas regeneration source protein comprises a human pancreas regeneration source protein REG3A shown in SEQ ID NO.1 and a murine pancreas regeneration source protein RegIII gamma shown in SEQ ID NO. 2. Wherein the pancreatic regeneration-derived protein derivatives comprise human pancreatic regeneration-derived protein derivatives C-REG3A shown in SEQ ID NO.3 and murine pancreatic regeneration-derived protein derivatives C-RegIII gamma shown in SEQ ID NO. 4.

The invention also provides application of the pancreatic regeneration protein derivative in preparation of bactericidal drugs and anti-infective drugs. The pancreatic regeneration source protein derivative can be used as a bactericide. The pancreatic regeneration-derived protein derivative can be used for preparing anti-infective drugs. Wherein the pancreatic regeneration-derived protein derivatives comprise human pancreatic regeneration-derived protein derivatives C-REG3A shown in SEQ ID NO.3 and murine pancreatic regeneration-derived protein derivatives C-RegIII gamma shown in SEQ ID NO. 4.

The invention also provides a composition or a kit, which contains the pancreatic regeneration source protein; the pancreatic regeneration source protein comprises human pancreatic regeneration source protein REG3A shown in SEQ ID NO.1 and/or murine pancreatic regeneration source protein RegIII gamma shown in SEQ ID NO. 2. The composition or kit may be used to effectively promote excessive inflammatory wound healing; can be used for effectively inhibiting excessive inflammation of diabetic skin wound and promoting diabetic wound healing; can be used as wound healing promoter or wound inflammation inhibitor in cosmetics and skin care products; can be used for inhibiting the expression of TNF-alpha and IL-6, and inhibiting inflammatory reaction induced by Poly (I: C).

The invention also proposes a composition or kit comprising a pancreatic regenerating-derived protein derivative; the pancreatic regeneration-derived protein derivatives comprise human pancreatic regeneration-derived protein derivatives C-REG3A shown in SEQ ID NO.3 and murine pancreatic regeneration-derived protein derivatives C-RegIII gamma shown in SEQ ID NO. 4. The composition or kit can be used for inhibiting inflammatory response, promoting wound healing, and resisting infection; can be used for inhibiting excessive inflammation of skin wound; can be used as wound healing promoter, wound inflammation inhibitor, and anti-infective agent in cosmetics or skin care products; can be used as anti-infective drug; can be used as a bactericide; can be used for inhibiting inflammatory reaction induced by Poly (I: C).

The composition comprises an effective amount of the pancreatic regeneration-derived protein and/or the pancreatic regeneration-derived protein derivative, and one or more of any pharmaceutically acceptable carriers, excipients, auxiliaries and the like, including but not limited to diluents, binders, wetting agents and the like. It can be made into any dosage form suitable for human or animal, including but not limited to injection, liniment, sustained release agent, etc. It can be prepared according to a known method. The content of the pancreatic regeneration-derived protein or the pancreatic regeneration-derived protein derivative in the composition or the kit is 0.1 to 99% by weight. In particular embodiments, the composition is: for example, the pancreatic regeneration-derived protein and its derivatives are diluted with a phosphate buffer (pH6.5, 20 mM). For example, an injection of the pancreatic regeneration-derived protein and its derivatives is prepared by mixing 100ug of the protein with 100ul of phosphate buffer (pH6.5, 20 mM). For example, the derivative of a pancreatic regenerating-derived protein is prepared in MES solution (MES: 25mM, NaCl: 25mM, pH: 6.0).

The invention provides the application of the pancreatic regeneration protein and the pancreatic regeneration protein derivative in inhibiting skin wound inflammation for the first time. The invention firstly provides that the pancreas regeneration source protein and the pancreas regeneration source protein derivative can reduce the inflammatory reaction of the diabetic wound and effectively inhibit the excessive inflammatory reaction of the skin wound, thereby promoting the healing of the diabetic wound. The invention discloses pancreas regeneration source protein and pancreas regeneration source protein derivatives with the functions of sterilizing and promoting wound healing. The invention provides a pancreas regeneration source protein and a pancreas regeneration source protein derivative, which not only can inhibit wound inflammation and promote the wound healing of diabetes, but also have bactericidal effect and have excellent application prospect.

Drawings

FIG. 1A shows REG3A protein expression;

FIG. 1B shows REG3A protein purification;

FIG. 2A shows RegIII γ protein expression;

FIG. 2B shows RegIII γ protein purification;

FIG. 3A shows C-REG3A protein expression;

FIG. 3B shows C-REG3A protein purification;

FIG. 4A shows C-RegIII γ protein expression;

FIG. 4B shows C-RegIII gamma protein purification;

FIG. 5A shows that REG3A inhibits poly (I: C) -induced mRNA expression of TNF-. alpha.;

FIG. 5B shows that REG3A inhibits poly (I: C) -induced mRNA expression of IL-6;

FIG. 6A shows that RegIII γ inhibits poly (I: C) -induced TNF- α mRNA expression;

FIG. 6B shows that RegIII γ inhibits poly (I: C) -induced mRNA expression of IL-6;

FIG. 7A shows that RegIII γ promotes diabetic skin wound healing;

FIG. 7B shows the healing curve for RegIII γ promoting healing of diabetic skin wounds;

FIG. 7C shows that RegIII γ inhibits the mRNA expression of TNF- α in diabetic skin wounds;

FIG. 7D shows that RegIII γ inhibits the mRNA expression of IL-6 in diabetic skin wounds;

FIG. 8A shows that REG3A promotes wound healing in diabetic skin;

FIG. 8B is a statistical chart showing that REG3A promotes wound healing in diabetic skin;

FIG. 8C shows that REG3A inhibited the mRNA expression of TNF- α in diabetic skin wounds;

FIG. 9A shows that C-REG3A inhibits poly (I: C) -induced mRNA expression of TNF-. alpha.;

FIG. 9B shows that C-REG3A inhibits poly (I: C) -induced mRNA expression of IL-6;

FIG. 10A shows that C-RegIII γ inhibits poly (I: C) -induced TNF- α mRNA expression;

FIG. 10B shows that C-RegIII γ inhibits poly (I: C) -induced mRNA expression of IL-6;

FIG. 11A shows that C-REG3A promotes skin wound healing;

FIG. 11B shows the healing curves of C-REG3A promoting skin wound healing;

FIG. 12A shows that C-RegIII γ promotes skin wound healing;

FIG. 12B shows the healing curve of C-RegIII γ promoting skin wound healing;

FIG. 13A shows the bactericidal effect of C-REG 3A;

FIG. 13B shows the bactericidal effect of C-RegIII γ.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1: preparation of pancreatic regeneration-derived protein REG3A/RegIII γ and its derivative C-REG3A/C-RegIII γ

The same method for preparing pancreatic regeneration-derived protein REG3A/RegIII γ and its derivative C-REG3A/C-RegIII γ is used, and the preparation of pancreatic regeneration-derived protein REG3A is taken as an example below:

(1) amplifying by using a PCR method to obtain a target gene, carrying out restriction enzyme digestion, cloning the target gene into an escherichia coli expression vector pET-32a (+), and inducing by IPT6(1mM) for 4h to express the pancreatic regeneration source protein REG 3A;

(2) collecting thallus, performing ultrasonic bacteria breaking, and allowing human pancreas regeneration source protein REG3A to exist in the precipitate in the form of inclusion body, as shown in 5 of figure 1A;

(3) renaturing the inclusion body after denaturation, and removing salt ions by using a dialysis bag;

(4) purifying the dialyzed protein by using a cation column, performing ultrafiltration concentration to obtain pancreas regeneration-derived protein REG3A, wherein as can be seen from FIG. 1B, most of pancreas regeneration-derived protein REG3A is eluted from 200mM NaCl solution;

(5) collecting 200mM NaCl eluate, dialyzing to remove salt ions, and removing endotoxin by anion column;

(6) ultrafiltering, concentrating, and freezing at-80 deg.C.

FIG. 1 shows the purification of REG3A protein expression; FIG. 2 shows the expression purification of the RegIII γ protein; FIG. 3 shows the expression purification of C-REG3A protein; FIG. 4 shows the expression purification of C-RegIII gamma protein.

Example 2: pancreatic regeneration-derived protein REG3A/RegIII gamma and derivative C-REG3A/C-RegIII gamma thereof inhibit Poly (I: C) -induced inflammatory response in vitro

Inoculating primary cultured human keratinocytes into a 24-well plate, adding pancreatic regeneration eggs REG3A, RegIII gamma, pancreatic regeneration egg derivatives C-REG3A and C-RegIII gamma 0.03nM in advance when the cells grow to 70% -80% full, adding 5 mu g/ml Poly (I: C) after 10min, extracting RNA or protein after 24h, and detecting the expression of TNF-alpha and IL 6.

The results of the experiments are shown in FIGS. 5A, 5B, 6A, 6B, 9A, 9B, 10A, 10B, and show that poly (I: C) can induce the expression of TNF-alpha and IL-6 in large quantities; the addition of pancreatic regeneration-derived protein REG3A/RegIII gamma or pancreatic regeneration-derived protein derivatives C-REG3A and C-RegIII gamma can inhibit poly (I: C) -induced TNF-alpha and IL-6 expression. Furthermore, the inhibitory effect of the pancreatic regeneration-derived protein derivatives C-REG3A and C-RegIII γ was significantly stronger than that of the pancreatic regeneration-derived proteins REG3A and RegIII γ (. beta.P < 0.05,. beta.P < 0.01,. beta.P < 0.001).

Example 3: the pancreas regeneration-derived protein REG3A or RegIII gamma inhibits the inflammatory reaction of the diabetic wound and promotes the wound healing of the diabetic mouse.

Step 1: c57BL/6 wild type mice were injected with Streptozotocin (STZ) at 50mg/kg by body weight for 5 consecutive days. After the mice are stabilized for 10 days, the blood sugar of the mice is detected, and the mice with the random blood sugar value larger than 16mmol/L are regarded as the success of the model building of the diabetic mice.

Step 2: diabetic mice were divided into two groups 24 hours after depilation, diabetic mouse control group: injection of PB, diabetic mice experimental group: 100 μ g of REG3A or RegIII γ was injected subcutaneously. After 6 hours, a wound having a diameter of 8mm was made at the injection site, and on the third day, tissues around the mouse wound were excised, RNA or protein was extracted, and the expression of TNF-. alpha.and IL-6 was examined.

When observing the time curve of the wound healing of the mouse, the area of the wound is photographed and recorded every day, and the wound healing speed is statistically analyzed.

The experimental results are shown in fig. 7A and 7B and fig. 8A and 8B: both REG3A and RegIII γ promoted wound healing in diabetic mice. As shown in fig. 7C, 7D and 8C: elevated expression of TNF- α and IL-6 at the diabetic wound as compared to expression of TNF- α and IL-6 in skin distal to the wound site; both the pancreatic regeneration-derived proteins REG3A and RegIII γ were effective in reducing the expression of TNF- α or IL-6 in wounds of diabetic mice, indicating that REG3A and RegIII γ were effective in inhibiting the inflammatory response of diabetic wounds in vivo, and further promoting the healing of diabetic wounds (. beta.P < 0.05,. beta.P < 0.01,. beta.P < 0.001).

Example 4: pancreas regeneration source protein derivatives C-REG3A and C-RegIII gamma function for promoting wound healing of mice

C57 normal mice were divided into three groups 24 hours after dehairing, normal mouse control group: injecting PB; normal mouse experimental group: subcutaneous injections of 100. mu. g C-REG3A or REG 3A. After 6 hours, a wound having a diameter of 8mm was prepared at the injection site, the area of the wound was recorded by photographing every day, and the healing rate of the wound was statistically analyzed. As shown in FIGS. 11A and 11B, C-REG3A was effective in promoting wound healing in mice (. SP., < 0.05). C-REG3A has the same wound healing promoting ability as REG 3A.

The example of C-RegIII γ promoting wound healing was the same as that of C-REG3A, and C-RegIII γ was also effective in promoting wound healing in mice (P < 0.05), as shown in 12A and 12B. C-RegIII γ has the same ability to promote wound healing as RegIII γ.

Example 5: sterilization effect of pancreatic regeneration source protein derivatives C-REG3A and C-RegIII gamma

The first step is as follows: bacterial Collection

1) The staphylococcus aureus is streaked on a solid culture medium TSA, after the staphylococcus aureus grows out, a single colony is picked and inoculated in a liquid culture medium TSB, and the staphylococcus aureus is cultured overnight at 37 ℃ and 220rpm in a shaking way. 2) The overnight-cultured activated bacteria were inoculated at 4% into fresh medium, cultured with shaking at 220rpm at 37 ℃ and the OD600 value of the bacterial growth was measured at various times. 3) When OD600 is 0.6-0.8, the cells were collected. The membrane-sterilized MES solution (MES 2-morpholino-ethanesulfonic acid: 25mM, NaCl: 25mM, pH: 6.0) was resuspended, centrifuged at 5000rpm for 5min, and the supernatant was discarded. This operation was repeated twice. 4) The OD600 values of Staphylococcus aureus were determined using MES solution as a blank.

The second step is that: bacterial incubation

1) Preparing an incubation system of staphylococcus aureus: 1 × 10⁵A CFU; C-REG3A/C-RegIII γ: 0uM, 2uM, 4uM, 6uM, 8 uM; the volume is adjusted to 100uL by MES.

2) After staphylococcus aureus was mixed with C-REG3A or C-RegIII γ, respectively, using a pipette gun, the samples were incubated in a 37 ℃ bacterial incubator for 3h at constant temperature.

3) And (6) pointing a board. The bacterial incubation system was subjected to gradient dilutions (1: 10, 1: 100, 1: 1000, 1: 10000, etc.), each dilution concentration was spotted on TSA solid medium, each set of triplicates, each replicate being a 10uL dilution sample.

4) The culture plate is placed upside down in a bacteria incubator at 37 ℃ for constant temperature culture for 16-20 h.

5) When a single colony grew on the plate and was clearly visible, it was counted. The number of bacteria in the stock solution was counted and analyzed for the inhibition rate of C-REG3A or C-RegIII γ.

The results of the experiment are shown in FIG. 13: FIG. 13A shows the bactericidal activity effect of C-REG3A, and FIG. 13B shows that the bactericidal activity effects of C-RegIII γ are all significantly different (. about.P < 0.001), indicating that C-REG3A/C-RegIII γ all have bactericidal activity, i.e., the bactericidal activity of REG3A/RegIII γ is retained.

The pancreatic regeneration-derived protein and its derivatives used in example 2 were prepared by diluting with a phosphate buffer (pH6.5, 20 mM). The injections of the pancreatic regeneration-derived protein and derivatives thereof used in examples 3 and 4 were prepared from 100ug of the protein and 100ul of phosphate buffer (pH6.5, 20 mM). The trypsin-derived protein derivative bactericide used in example 5 was prepared from a MES solution (MES: 25mM, NaCl: 25mM, pH 6.0).

Claims

1. The application of the pancreatic regeneration-derived protein in preparing the medicine for promoting the healing of the excessive inflammatory wound is characterized in that the pancreatic regeneration-derived protein is human pancreatic regeneration-derived protein REG3A shown in SEQ ID NO.1 or murine pancreatic regeneration-derived protein RegIII gamma shown in SEQ ID NO. 2; the medicine utilizes the pancreatic regeneration source protein to inhibit excessive inflammatory reaction to promote wound healing; wherein the pancreatic regeneration-derived protein promotes the healing of excessive inflammatory wounds by inhibiting inflammatory factors.

2. The application of the pancreas regeneration source protein in preparing the medicine for promoting the healing of the diabetes excessive inflammation wound; wherein the pancreatic regeneration source protein is human pancreatic regeneration source protein REG3A shown in SEQ ID NO.1 or murine pancreatic regeneration source protein RegIII gamma shown in SEQ ID NO. 2; the pancreas regeneration source protein inhibits excessive inflammation of the skin wound of the diabetes and promotes the healing of the wound of the diabetes by inhibiting inflammatory factors.

3. The pancreatic regeneration source protein is used as an excessive inflammation wound healing promoter or an excessive wound inflammation inhibitor in cosmetics and skin care products; wherein the pancreas regeneration source protein comprises a human pancreas regeneration source protein REG3A shown in SEQ ID NO.1 and a murine pancreas regeneration source protein RegIII gamma shown in SEQ ID NO. 2; wherein the pancreatic regeneration-derived protein promotes excessive inflammatory wound healing or inhibits excessive wound inflammation by inhibiting inflammatory factors.

4. The use of any one of claims 1 to 3, wherein the pancreatic regenerating-derived protein inhibits the expression of the periwound inflammatory factors TNF- α and IL-6.

5. The use of claim 2, wherein the diabetic hyper-inflammatory wound is a diabetic foot ulcer.